CN112362768B - Method for measuring Berol 185 content in sample by utilizing liquid chromatography-tandem mass spectrometry - Google Patents
Method for measuring Berol 185 content in sample by utilizing liquid chromatography-tandem mass spectrometry Download PDFInfo
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- ZBJVLWIYKOAYQH-UHFFFAOYSA-N naphthalen-2-yl 2-hydroxybenzoate Chemical compound OC1=CC=CC=C1C(=O)OC1=CC=C(C=CC=C2)C2=C1 ZBJVLWIYKOAYQH-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 59
- 150000002500 ions Chemical class 0.000 claims abstract description 47
- 239000000126 substance Substances 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 108
- 239000012086 standard solution Substances 0.000 claims description 35
- 238000004949 mass spectrometry Methods 0.000 claims description 20
- 238000001819 mass spectrum Methods 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 229940066827 pertussis vaccine Drugs 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000004811 liquid chromatography Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000007781 pre-processing Methods 0.000 claims description 2
- 238000005173 quadrupole mass spectroscopy Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 16
- 238000004458 analytical method Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 69
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 7
- 238000011068 loading method Methods 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- 238000004364 calculation method Methods 0.000 description 6
- 238000011835 investigation Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 238000013375 chromatographic separation Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000000611 regression analysis Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102000004310 Ion Channels Human genes 0.000 description 3
- 201000005702 Pertussis Diseases 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108010081690 Pertussis Toxin Proteins 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002736 nonionic surfactant Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
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Abstract
The invention provides a method for measuring the Berol 185 content in a sample by utilizing liquid chromatography-tandem mass spectrometry, which comprises the following steps: A. preparing Berol 185 standard substance solutions with different concentrations, and performing liquid chromatography tandem mass spectrometry detection; screening out quantitative ions according to the detection result of the liquid chromatography tandem mass spectrometry; B. respectively extracting peak areas of quantitative ion peaks of standard substance solutions with different concentrations, and drawing a peak area-concentration standard curve; C. pretreating a sample, and then adopting the method in A to perform liquid chromatography tandem mass spectrometry detection; D. and according to the detection result of the sample, comparing the standard curve to obtain the concentration of the Berol 185 in the sample. The method has the advantages of simple sample pretreatment, quick sample introduction, good sensitivity, high recovery rate, good precision and repeatability, and can realize the purpose of simple, quick and accurate analysis of the Berol 185 content.
Description
Technical Field
The invention relates to the field of biological product detection, in particular to a method for measuring the Berol 185 content in a sample by utilizing liquid chromatography-tandem mass spectrometry.
Background
Berol 185, chemical name C10-16 fatty alcohol polyoxyethylene polyoxypropylene ether, molecular formula is R-O- (C) 2 H 4 O)n-(C 3 H 6 O)m-H：R＝C n H 2n+1 (n=10-16), which is a hydrophilic nonionic surfactant, has no ultraviolet absorption, no fluorescence, and is a clear liquid, and has a pH range of 5-7 in 1% water, and is soluble in water, isopropanol, methanol, and ethanol.
On the one hand, in the production process of the pertussis vaccine, the Berol 185 has remarkable effect on controlling the endotoxin content after the surfactant is added; berol 185, on the other hand, is toxic to aquatic organisms. Therefore, establishing a method for rapidly, accurately, sensitively and reliably detecting the residual quantity of Berol 185 in a vaccine is particularly important for safe production of the vaccine. The liquid chromatography-mass spectrometry technology is a separation analysis technology which takes high performance liquid chromatography as a separation means and mass spectrometry as an identification tool. At present, no relevant report on the application of the LC-MS/MS technology to the analysis or detection of the Berol 185 content is seen.
Disclosure of Invention
The invention aims to provide a method for measuring the Berol 185 content in a sample by utilizing liquid chromatography-tandem mass spectrometry.
To achieve the object of the present invention, in a first aspect, the present invention provides a method for determining the Berol 185 content in a sample by using liquid chromatography tandem mass spectrometry, comprising the steps of:
A. preparing Berol 185 standard substance solutions with different concentrations, and performing liquid chromatography tandem mass spectrometry detection; screening out quantitative ions according to the detection result of the liquid chromatography tandem mass spectrometry;
determining Retention Time (RT) according to peak positions in a mass spectrogram, wherein RT values of standard substance solutions with different concentrations are the same;
B. respectively extracting quantitative ion peaks of standard substance solutions with different concentrations and recording peak areas; drawing a standard curve by taking the concentration of the standard solution as an abscissa and the peak area corresponding to the quantitative ions as an ordinate;
C. pretreating a sample to be detected, and then adopting the same method as the step A to carry out liquid chromatography tandem mass spectrometry detection;
D. and according to the detection result of the sample to be detected, comparing the standard curve to obtain the concentration of the Berol 185 in the sample to be detected, thereby realizing quantitative detection of the Berol 185 in the sample.
In the method, in the step A, the solution of the Berol 185 standard substance with different concentrations is prepared by methanol.
The method for preprocessing the sample to be detected in the step C comprises the following steps: mixing a sample to be detected with methanol according to the volume ratio of 1:2-1:9, centrifuging, and taking supernatant for liquid chromatography tandem mass spectrometry detection.
Preferably, the liquid chromatography uses a Bio C18 reverse-phase chromatography column (GL Science Bio C18 chromatography column, 100X 2.1mm,1.7 μm); the mobile phase A is formic acid aqueous solution with the volume percentage of 0.1%; mobile phase B is methanol; the flow rate is 0.25-0.35ml/min; the column temperature is 25-35 ℃; the sample amount is preferably 10. Mu.L.
Gradient elution is adopted: 0.00-1.00min 5% B (the rest of the components are mobile phase A), 2.01-5.00min 40% B (the rest of the components are mobile phase A), 5.01-7.00min 99% B (the rest of the components are mobile phase A), 7.01-9.00min99% B (the rest of the components are mobile phase A), 9.01-9.10min 40% B (the rest of the components are mobile phase A), 9.11-15.00min 5% B (the rest of the components are mobile phase A).
Preferably, the mass spectrometry detection conditions are as follows:
performing mass spectrum detection by using a triple quadrupole mass spectrometry system; an ESI ionization source; a positive ion detection mode; atomizing air flow rate: 2.5-3L/min; interface temperature: 250-350 ℃; DL temperature: 200-280; heating block temperature: 350-450 ℃.
In the present invention, the parent ion of the detection signal generated by the Berol 185 standard in mass spectrometry has a value of 630.05 (m+nh 4 ) The sub-ions comprise sub-ions having mass to charge ratios 89.10, 133.10 and 613.40. Wherein parent ion 630.05 and child ion 89.10 can be used as quantitative ion pairs, and parent ion 630.05 and child ion 613.40, and child ion 133.10 can be used as two qualitative ion pairs.
In the method, the linear detection range of Berol 185 in the sample is 5-120ppb, and the detection limit is 1ppb.
In the present invention, the sample includes, but is not limited to, biological products, preferably vaccine products, more preferably vaccines containing pertussis antigens.
In a second aspect, the invention provides the use of the above method for quality control of biological products, in particular vaccine products comprising pertussis antigens.
The vaccine containing the pertussis antigen refers to any one or combination of Filiform Hemagglutinin (FHA) and Pertussis Toxin (PT) in vaccine components.
The invention establishes a method for analyzing Berol 185 by using a liquid chromatography-mass spectrometer for the first time. Berol 185 is a polymer, and has relatively complex molecular structure, large molecular weight and low polarity, and quantitative detection of trace Berol 185 is difficult to realize by using a general detection method. The invention establishes a method for detecting the Berol 185 content in the sample through systematic research and a large number of experimental verification. The method has at least the following advantages:
the liquid chromatography-mass spectrometry has the characteristics of high separation degree of the chromatograph and high specificity and high sensitivity of the chromatograph, so that the research on the quantitative test of the sample Berol 185 can be realized rapidly and accurately;
the pretreatment method is simple and convenient to operate, so that the analysis of the Berol 185 content in the sample is realized simply and rapidly;
the extraction reagent used in the invention is methanol, and has low cost, little pollution and little toxicity;
the invention has the characteristics of good precision, high recovery rate and good repeatability, and the detection limit can reach 1ppb.
Drawings
FIGS. 1 a-1 c are mass spectra of a Berol 185 liquid chromatography-mass spectrometry assay in a sample according to a preferred embodiment of the present invention; wherein, fig. 1a: a blank sample mass spectrum; fig. 1b: adding a Berol 185 standard quality spectrogram into the blank; fig. 1c: and (5) actually measuring a mass spectrum of the sample.
FIG. 2 is a mass spectrum of a sample measured in accordance with a preferred embodiment of the present invention, and an ion spectrum extracted from the peak.
Fig. 3 a-3 c show the Berol 185 standard curve in the preferred embodiment of the present invention.
Detailed Description
The invention provides a liquid chromatography-mass spectrometry detection method of a Berol 185 nonionic surfactant. The method comprises the following specific steps:
1. pretreating a sample of an object to be detected containing Berol 185, taking the sample to be detected (such as pertussis vaccine), adding methanol into the sample to be detected to carry out protein precipitation, wherein the volume ratio of the sample to be detected to the methanol is 1:4, and taking supernatant after centrifugation as a detection sample.
2. Chromatographic separation
The chromatographic column is a Bio C18 reverse chromatographic column; the mobile phase A is formic acid aqueous solution with the volume percentage of 0.1%; mobile phase B is methanol; flow rate: 0.3ml/min; the temperature of the column temperature box is 35 ℃; the chromatographic gradient elution conditions were: 0.00-1.00min 5%B,2.01min-5.00min 40%B,5.01-7.00min 99%B,7.01-9.00min 99%B,9.01-9.10min 40%B,9.11-15.00min 5% B, and the rest components are A; the sample loading was 10. Mu.L.
3. Mass spectrometry detection
An ESI ionization source; a positive ion detection mode; atomizing air flow rate: 2.5-3L/min; interface temperature: 250-350 ℃; DL temperature: 200-280 ℃; heating block temperature: 350-450 ℃; quantitative ion: parent 630.05 and child 89.10, ce: -30.0V, qualitative ion: parent 630.05 and child 613.45, ce: -18.0V, parent 630.05 and child 133.10, ce: -26.0V.
4. Drawing of a Standard Curve
Standard solutions of different concentrations of Berol 185 were prepared separately and were assayed using the chromatographic and mass spectrometric conditions described above. The method comprises the following steps:
weighing 20mg Berol 185, adding 10ml methanol to a volumetric flask of 10ml to fix the volume, swirling until the sample is sufficiently dissolved to obtain a primary standard solution with the concentration of 2000ppm, taking 50 mu L of the primary standard solution, adding the primary standard solution into a volumetric flask of 100ml, and adding the methanol to fix the volume to 100ml to obtain a secondary standard solution of 1ppm; respectively taking 50, 100, 200, 400, 600, 800, 1000, 1200 mu L of secondary standard solution into 10ml volumetric flasks, and respectively metering the volume to 10ml by methanol to obtain 5, 10, 20, 40, 60, 80, 100. Measuring 120ppb of serial standard liquids in 1.5ml sample bottles, adopting the chromatographic and mass spectrometry conditions to measure the standard liquids with different concentrations, respectively extracting quantitative ion peaks of the standard liquids with different concentrations, recording peak areas, and drawing peak area-concentration standard curves; regression analysis is carried out according to the peak area of the extracted ions corresponding to the quantitative ions and the concentrations of different standard solutions, so as to obtain a linear equation: y=11579.8x+47832.2, r 2 = 0.9986953; where x is the Berol 185 concentration (ppb) and y is the extracted ion peak area.
5. And (5) examining the recovery rate.
6. And (5) examining precision.
7. Sample detection to be tested
Taking 2.0ml of a sample to be detected in a 15ml centrifuge tube, adding 8.0ml of methanol, swirling for 30s, taking 1.0ml of mixed solution in a 1.5ml centrifuge tube, centrifuging for 10min with 14000g, taking the supernatant in a 1.5ml sample injection bottle, and measuring the treated sample by adopting the chromatographic and mass spectrum conditions; and calculating the concentration of the target object through an equation obtained by a standard curve.
The liquid chromatography-mass spectrometry detection system used in the invention is an ultra-high performance liquid chromatography tandem triple quadrupole mass spectrum.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available. Example 1 method for determining the Berol 185 content in a sample Using liquid chromatography tandem Mass Spectrometry
1. Analytical instrument and reagent
Analytical instrument: the SHIMADZU-8050 triple quadrupole mass spectrometer is matched with an ESI electrospray ion source, and analysis software is a Labsolutions 5.93 data processing system; the Shimadzu LC20AD XR high performance liquid chromatograph is carried with a Shimadzu Nexera X2 autosampler; the column was a GL Science Bio C18 column (100X 2.1mm,1.7 μm).
The experimental reagents are shown in table 1:
TABLE 1
Note that: NA indicates inapplicability.
2. Experimental methods and results
2.1 pretreatment of Berol 185 containing samples
Taking 2.0ml of a sample to be detected in a 15ml centrifuge tube, adding 8.0ml of methanol, swirling for 30s, taking 1.0ml of mixed solution in a 1.5ml centrifuge tube, centrifuging for 10min at 14000g, taking the supernatant in a 1.5ml sample injection bottle, and loading for analysis.
2.2 chromatographic separation
A chromatographic column; GL Science Bio C18 column (100X 2.1mm,1.7 μm); mobile phase: a is formic acid aqueous solution with the volume percentage of 0.1 percent; b is 100% methanol; the flow rate is 0.3ml/min; the temperature of the column temperature box is 35 ℃; the chromatographic gradient elution conditions were: 0.00-1.00min 5%B,2.01min-5.00min 40%B,5.01-7.00min 99%B,7.01-9.00min 99%B,9.01-9.10min 40%B,9.11-15.00min 5% B; the rest components are A; the sample loading was 10. Mu.L.
2.3 Mass Spectrometry detection
Mass spectrometry conditions: ESI ionization source, positive ion detection mode, atomizing airflow: 3L/min, interface temperature: 300 ℃; DL temperature: 250 ℃, heating block temperature: ion channels at 400 ℃ are shown in table 2:
TABLE 2
2.4 establishment of standard curve
Measuring 20mg of Berol 185 (batch number: 1576960) standard substance in a 10ml volumetric flask, adding 10ml of methanol to fix volume, swirling until the sample is fully dissolved to obtain a primary standard solution with the concentration of 2000ppm, taking 50 mu L of the primary standard solution in a 100ml volumetric flask, adding methanol to fix volume to 100ml to obtain a secondary standard solution of 1ppm; respectively taking 50, 100, 200, 400, 600, 800, 1000 and 1200 mu L of secondary standard solution into 10ml volumetric flasks, respectively metering the volume to 10ml by methanol to obtain systems of 5, 10, 20, 40, 60, 80, 100 and 120ppbRespectively taking 1ml of standard solution into a 1.5ml sample injection bottle, measuring the standard solutions with different concentrations by adopting the chromatographic and mass spectrometry conditions, respectively extracting quantitative ion peaks with different concentrations, recording peak areas, and drawing a peak area-concentration standard curve; regression analysis (Table 3) is performed according to the peak areas of extracted ions corresponding to the quantitative ions and the concentrations of different objects to be detected, so as to obtain a linear equation: y=31175.8x+348341, r 2 = 0.9951452; where x is the Berol 185 concentration (ppb) and y is the extracted ion peak area.
The minimum limit value of the Berol 185 content measured by the method is 1ppb, and the linear detection range of the Berol 185 is 5-120ppb.
TABLE 3 Table 3
| Sample name | Retention time (min) | Peak area | Ppb concentration of |
| S1 | 9.378 | 488,806 | 4.506 |
| S2 | 9.384 | 664,491 | 10.141 |
| S3 | 9.388 | 917,781 | 18.265 |
| S4 | 9.39 | 1,511,772 | 37.318 |
| S5 | 9.39 | 2,349,253 | 64.182 |
| S6 | 9.383 | 2,977,844 | 84.344 |
| S7 | 9.394 | 3,473,185 | 100.233 |
| S8 | 9.384 | 3,965,054 | 116.01 |
2.5 investigation of accuracy and recovery
Taking the secondary standard solution 0 (control), 200, 400 and 600 mu L, respectively adding 2000 mu L of a sample (pertussis vaccine FHA refined solution (20200520)), respectively using methanol to fix the volume to 10ml, swirling for 30s, centrifuging 14000g for 10min, taking the supernatant in a 1.5ml sample bottle, respectively analyzing a sample matrix and four samples with different concentrations by adopting the chromatographic and mass spectrometry conditions, obtaining a detection content value according to a standard curve equation, and calculating the recovery rate according to the following formula:
the calculation results are shown in table 4:
TABLE 4 Table 4
Note that: NA indicates inapplicability.
2.6 precision investigation
Taking 400 mu L (5 parts) of the secondary standard solution, fixing the volume to 10ml by using methanol, swirling for 30 seconds, taking the supernatant in a 1.5ml sample bottle, respectively analyzing 5 samples by adopting the chromatographic and mass spectrometry conditions, obtaining a detection content value according to a standard curve equation, calculating the relative deviation of the 5 samples, and calculating the calculation result as shown in table 5:
TABLE 5
| Sample name | 1 | 2 | 3 | 4 | 5 | RSD(％) |
| Content (ppb) | 41.565 | 40.893 | 41.387 | 41.131 | 41.419 | 1％ |
2.7 conclusion
From the above results, the detection method regresses the equation R 2 Greater than 0.995, three-point labeling recovery is between 80% and 120%, and rsd=1%, which indicates that the method has good sensitivity, good precision and high recovery rate.
The mass spectrum of the Berol 185 liquid chromatography-mass spectrometry in the sample is shown in figures 1 a-1 c; wherein, fig. 1a: a blank sample mass spectrum; fig. 1b: adding a Berol 185 standard quality spectrogram into the blank; fig. 1c: and (5) actually measuring a mass spectrum of the sample.
The mass spectrum of the sample is actually measured, and the peak value highest point extraction ion spectrum is shown in figure 2.
The Berol 185 standard curves are shown in fig. 3 a-3 c, respectively.
Example 2 actual sample detection
1. Analytical instrument and reagent
Analytical instrument: the SHIMADZU-8050 triple quadrupole mass spectrometer is matched with an ESI electrospray ion source, and analysis software is a Labsolutions 5.93 data processing system; the Shimadzu LC20AD XR high performance liquid chromatograph is carried with a Shimadzu Nexera X2 autosampler; the column was a GL Science Bio C18 column (100X 2.1mm,1.7 μm).
The experimental reagents are shown in table 6:
TABLE 6
2. Experimental methods and results
2.1 pretreatment of Berol 185 containing samples
Taking 2.0ml of a sample to be detected in a 15ml centrifuge tube, adding 8.0ml of methanol, swirling for 30s, taking 1.0ml of mixed solution in a 1.5ml centrifuge tube, centrifuging for 10min at 14000g, taking the supernatant in a 1.5ml sample injection bottle, and loading for analysis.
2.2 chromatographic separation
A chromatographic column; GL Science Bio C18 column (100X 2.1mm,1.7 μm); mobile phase: a is formic acid aqueous solution with the volume percentage of 0.1 percent; b is 100% methanol; the flow rate is 0.3ml/min; the temperature of the column temperature box is 35 ℃; the chromatographic gradient elution conditions were: 0.00-1.00min 5%B,2.01min-5.00min 40%B,5.01-7.00min 99%B,7.01-9.00min 99%B,9.01-9.10min 40%B,9.11-15.00min 5% B; the rest components are A; the sample loading was 10. Mu.L.
2.3 Mass Spectrometry detection
Mass spectrometry conditions: ESI ionization source, positive ion detection mode, atomizing airflow: 3L/min, interface temperature: 300 ℃; DL temperature: 250 ℃, heating block temperature: 400 ℃; the ion channels are shown in table 7:
TABLE 7
2.4 establishment of standard curve
Measuring 20mg of Berol 185 (batch number: 1576973) standard substance in a 10ml volumetric flask, adding 10ml of methanol to fix volume, swirling until the sample is fully dissolved to obtain a primary standard solution with the concentration of 2000ppm, taking 50 mu L of the primary standard solution in a 100ml volumetric flask, adding methanol to fix volume to 100ml to obtain a secondary standard solution of 1ppm; respectively taking 50, 100, 200, 400, 600, 800, 1000 and 1200 mu L of secondary standard solution in a 10ml volumetric flask, respectively fixing the volume to 10ml by using methanol to obtain 5, 10, 20, 40, 60, 80, 100 and 120ppb of serial standard solutions, respectively taking 1ml of the standard solutions in a 1.5ml sample injection bottle, respectively measuring the standard solutions with different concentrations by adopting the chromatographic and mass spectrometry conditions, respectively extracting quantitative ion peaks with different concentrations, recording peak areas, and drawing a peak area-concentration standard curve; regression analysis (Table 8) is performed according to the peak areas of extracted ions corresponding to the quantitative ions and the concentrations of different objects to be detected, so as to obtain a linear equation: y= 35828.9x+321161，R 2 = 0.9950273; where x is the Berol 185 concentration (ppb) and y is the extracted ion peak area.
The minimum value of the Berol 185 content measured by the method is 1ppb. The linear detection range of Berol 185 is 5-120ppb;
TABLE 8
| Sample name | Retention time (min) | Peak area | Ppb concentration of |
| S1 | 9.316 | 497,536 | 4.923 |
| S2 | 9.312 | 656,612 | 9.363 |
| S3 | 9.302 | 974,863 | 18.245 |
| S4 | 9.300 | 1,687,199 | 38.127 |
| S5 | 9.297 | 2,592,997 | 63.408 |
| S6 | 9.307 | 3,330,267 | 83.986 |
| S7 | 9.304 | 3,976,510 | 102.022 |
| S8 | 9.296 | 4,438,858 | 114.927 |
2.5 investigation of accuracy and recovery
Taking the secondary standard solution 0 (control), 100, 200 and 300 mu L, respectively adding 2000 mu L of samples (pertussis vaccine FHA refined solution, batch No. 20200730), respectively using methanol to fix the volume to 10ml, centrifuging for 10min after swirling for 30s, taking the supernatant in a 1.5ml sample bottle, respectively analyzing a sample matrix and four samples with different concentrations by adopting the chromatographic and mass spectrometry conditions, obtaining detection content values according to a standard curve equation, and calculating the recovery rate according to the following formula:
the calculation results are shown in table 9:
TABLE 9
Note that: NA indicates inapplicability.
2.6 precision investigation
Taking 400 mu L (5 parts) of the secondary standard solution, fixing the volume to 10ml by using methanol, swirling for 30 seconds, taking the supernatant in a 1.5ml sample bottle, respectively analyzing 5 samples by adopting the chromatographic and mass spectrometry conditions, obtaining a detection content value according to a standard curve equation, calculating the relative deviation of the 5 samples, and calculating the calculation result as shown in table 10:
table 10
| Sample name | 1 | 2 | 3 | 4 | 5 | RSD(％) |
| Content (ppb) | 29.114 | 28.068 | 29.725 | 29.723 | 30.268 | 3％ |
2.7 conclusion
From the above results, the detection method regresses the equation R 2 Greater than 0.995, three-point labeling recovery is between 80% and 120%, and rsd=3%, which indicates that the method has good sensitivity, good precision and high recovery rate.
Example 3 actual sample detection
1. Analytical instrument and reagent
Analytical instrument: the SHIMADZU-8050 triple quadrupole mass spectrometer is matched with an ESI electrospray ion source, and analysis software is a Labsolutions 5.93 data processing system; the Shimadzu LC20AD XR high performance liquid chromatograph is carried with a Shimadzu Nexera X2 autosampler; the column was a GL Science Bio C18 column (100X 2.1mm,1.7 μm).
The experimental reagents are shown in table 11:
TABLE 11
Note that: NA indicates inapplicability.
2. Experimental methods and results
2.1 pretreatment of Berol 185 containing samples
Taking 2.0ml of a sample to be detected in a 15ml centrifuge tube, adding 8.0ml of methanol, swirling for 30s, taking 1.0ml of mixed solution in a 1.5ml centrifuge tube, centrifuging for 10min at 14000g, taking the supernatant in a 1.5ml sample injection bottle, and loading for analysis.
2.2 chromatographic separation
A chromatographic column; GL Science Bio C18 column (100X 2.1mm,1.7 μm); mobile phase: a is formic acid aqueous solution with the volume percentage of 0.1 percent; b is 100% methanol; the flow rate is 0.3ml/min; the temperature of the column temperature box is 35 ℃; the chromatographic gradient elution conditions were: 0.00-1.00min 5%B,2.01min-5.00min 40%B,5.01-7.00min 99%B,7.01-9.00min 99%B,9.01-9.10min 40%B,9.11-15.00min 5% B; the rest components are A; the sample loading was 10. Mu.L.
2.3 Mass Spectrometry detection
Mass spectrometry conditions: ESI ionization source, positive ion detection mode, atomizing airflow: 3L/min, interface temperature: 300 ℃; DL temperature: 250 ℃, heating block temperature: 400 ℃; the ion channels are shown in table 12:
table 12
2.4 establishment of standard curve
Measuring 20mg of Berol 185 (batch number: 1576978) standard substance in a 10ml volumetric flask, adding 10ml of methanol to fix volume, swirling until the sample is fully dissolved to obtain a primary standard solution with the concentration of 2000ppm, taking 50 mu L of the primary standard solution in a 100ml volumetric flask, adding methanol to fix volume to 100ml to obtain a secondary standard solution of 1ppm; respectively taking 50, 100, 200, 400, 600, 800, 1000 and 1200 mu L of secondary standard solution in a 10ml volumetric flask, respectively fixing the volume to 10ml by using methanol to obtain 5, 10, 20, 40, 60, 80, 100 and 120ppb of serial standard solutions, respectively taking 1ml of the standard solutions in a 1.5ml sample injection bottle, respectively measuring the standard solutions with different concentrations by adopting the chromatographic and mass spectrometry conditions, respectively extracting quantitative ion peaks with different concentrations, recording peak areas, and drawing a peak area-concentration standard curve; regression analysis (Table 13) was performed according to the peak areas of extracted ions corresponding to the quantitative ions and the concentrations of different analytes to obtain a linear equation: y=32684.6x+419135, r 2 = 0.9961489; where x is the Berol 185 concentration (ppb) and y is the extracted ion peak area.
The minimum limit value of the Berol 185 content measured by the method is 1ppb, and the linear detection range of the Berol 185 is 5-120ppb.
TABLE 13
| Sample name | Retention time (min) | Peak area | Ppb concentration of |
| S1 | 9.318 | 557,300 | 4.227 |
| S2 | 9.31 | 723,156 | 9.302 |
| S3 | 9.312 | 975,404 | 17.019 |
| S4 | 9.319 | 1,785,453 | 41.803 |
| S5 | 9.322 | 2,485,221 | 63.213 |
| S6 | 9.322 | 3,120,307 | 82.643 |
| S7 | 9.327 | 3,721,928 | 101.05 |
| S8 | 9.321 | 4,202,125 | 115.742 |
2.5 investigation of accuracy and recovery
Taking the secondary standard solution 0 (control), 100, 500 and 900 mu L, respectively adding 2000 mu L of samples (pertussis vaccine FHA refined solution, batch number: 20200925), respectively fixing the volume to 10ml by using methanol, centrifuging for 10min after swirling for 30s, taking the supernatant in a 1.5ml sample bottle, respectively analyzing a sample matrix and four samples with different concentrations by adopting the chromatographic and mass spectrometry conditions, obtaining detection content values according to a standard curve equation, and calculating the recovery rate according to the following formula:
the calculation results are shown in Table 14:
TABLE 14
Note that: NA indicates inapplicability.
2.6 precision investigation
200 mu L (5 parts) of the secondary standard solution is taken, methanol is used for fixing the volume to 10ml, the supernatant is taken in a 1.5ml sample injection bottle after 30s of vortex, the 5 samples are respectively analyzed by adopting the chromatographic conditions and the mass spectrum conditions, the detection content value is obtained according to a standard curve equation, the relative deviation of the 5 samples is calculated, and the calculation result is shown in table 15:
TABLE 15
| Sample name | 1 | 2 | 3 | 4 | 5 | RSD(％) |
| Content (ppb) | 20.131 | 20.939 | 20.326 | 20.552 | 19.085 | 3％ |
2.7 conclusion
From the above results, the detection method regresses the equation R 2 Greater than 0.995, three-point labeling recovery is between 80% and 120%, and RSD=3%, which indicates that the method has good sensitivity, good precision and high recovery rate.
In conclusion, the method for analyzing the Berol 185 content by liquid chromatography-mass spectrometry provided by the invention has the advantages of simple pretreatment, good precision, high recovery rate, good repeatability and low detection limit, and can be used for simply, rapidly and accurately analyzing the Berol 185 content in a sample.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (2)
1. A method for determining the Berol 185 content in a sample by using liquid chromatography tandem mass spectrometry, comprising the following steps:
A. preparing Berol 185 standard substance solutions with different concentrations by using methanol, and performing liquid chromatography tandem mass spectrometry detection; screening out quantitative ions according to the detection result of the liquid chromatography tandem mass spectrometry;
B. respectively extracting quantitative ion peaks of standard substance solutions with different concentrations, and recording peak areas; drawing a standard curve by taking the concentration of the standard solution as an abscissa and the peak area corresponding to the quantitative ions as an ordinate;
C. pretreating a sample to be detected, and then adopting the same method as the step A to carry out liquid chromatography tandem mass spectrometry detection;
D. according to the detection result of the sample to be detected, comparing the standard curve to obtain the concentration of the Berol 185 in the sample to be detected, thereby realizing quantitative detection of the Berol 185 in the sample;
the method for preprocessing the sample to be detected in the step C comprises the following steps: mixing a sample to be detected with methanol according to the volume ratio of 1:2-1:9, centrifuging, and taking supernatant for liquid chromatography tandem mass spectrometry detection;
the liquid chromatography adopts a Bio C18 reverse chromatography column; the mobile phase A is formic acid aqueous solution with the volume percentage of 0.1%; mobile phase B is methanol; the flow rate is 0.25-0.35ml/min; the column temperature is 25-35 ℃;
gradient elution is adopted: 0.00-1.00min 5% B,2.01-5.00min 40%B,5.01-7.00min 99%B,7.01-9.00min 99%B,9.01-9.10min 40%B,9.11-15.00min 5% B;
the mass spectrum detection conditions were as follows:
mass spectrometry detection was performed using a triple quadrupole mass spectrometry system: an ESI ionization source; a positive ion detection mode; atomizing air flow rate: 2.5-3L/min; interface temperature: 250-350 ℃; DL temperature: 200-280 ℃; heating block temperature: 350-450 ℃; quantitative ion: parent 630.05 and child 89.10, ce: -30.0V, qualitative ion: parent 630.05 and child 613.45, ce: -18.0V, parent 630.05 and child 133.10, ce: -26.0V; the sample is pertussis vaccine FHA refined solution.
2. The method of claim 1, wherein the Berol 185 in the sample has a linear detection range of 5 to 120ppb and a detection limit of 1ppb.
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